Cytokinin-induced activation of polyribosomes in the protonema of Ceratodon purpureus

A ribosomal preparation from N⁶-isopentenyladenine-treated protonema of Ceratodon purpureus (Hedw.) Brid. exhibited an increased activity of protein synthesis in a cell-free system as compared to a control preparation. The ratio of polyribosomes to monoribosomes was the same in both preparations, and it is assumed that an activation of pre-existing polyribosomes was responsible for the increased efficiency in protein synthesis. An electrophoretic fractionation of the in vitro translation product showed an enhanced synthesis of some polypeptide fractions in the cytokinin variant.     

Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.     

Eukaryotic Release Factor 3 Is Required for Multiple Turnovers of Peptide Release Catalysis by Eukaryotic Release Factor 1

Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.     

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.     

Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.     

Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site

Overexpression and activation of the c-Src protein have been linked to the development of a wide variety of cancers. The molecular mechanism(s) of c-Src overexpression in cancer cells is not clear. We report here an internal ribosome entry site (IRES) in the c-Src mRNA that is constituted by both 5′-noncoding and -coding regions. The inhibition of cap-dependent translation by m⁷GDP in the cell-free translation system or induction of endoplasmic reticulum stress in hepatoma-derived cells resulted in stimulation of the c-Src IRES activities. Sucrose density gradient analyses revealed formation of a stable binary complex between the c-Src IRES and purified HeLa 40 S ribosomal subunit in the absence of initiation factors. We further demonstrate eIF2-independent assembly of 80 S initiation complex on the c-Src IRES. These features of the c-Src IRES appear to be reminiscent of that of hepatitis C virus-like IRESs and translation initiation in prokaryotes. Transfection studies and genetic analysis revealed that the c-Src IRES permitted initiation at the authentic AUG351, which is also used for conventional translation initiation of the c-Src mRNA. Our studies unveiled a novel regulatory mechanism of c-Src synthesis mediated by an IRES element, which exhibits enhanced activity during cellular stress and is likely to cause c-Src overexpression during oncogenesis and metastasis.     

Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.     

Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis through Reduced ATPase Activity

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs.     

Mitotic modulation of translation elongation factor 1 leads to hindered tRNA delivery to ribosomes

Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.     

Translational signals of a major head protein gene of bacteriophage lambda

Summary The D gene of bacteriophage which codes for a major head protein is expressed at a high level during growth. We have constructed a set of D-lacZ gene fusions in order to examine the factors determining the high efficiency of the D translational initiation signals. It was found that an integral sequence, 300 bp long and upstream of the ATG initiation codon, is required for maximal protein synthesis.     

The role of tRNA as a molecular spring in decoding, accommodation, and peptidyl transfer

Translation is the process by which the genetic information contained in mRNA is used to link amino acids in a predetermined sequential order into a polypeptide chain, which then folds into a protein. Transfer RNAs (tRNAs) are the adapter molecules designed to provide the "lookup" from codons to amino acids. Cryo-EM has provided evidence that the ribosome, as a molecular machine, undergoes many structural changes during translation. Recent findings show that the tRNA structure itself undergoes large conformational changes as well, and that the decoding process must be seen as a complex dynamic interplay between tRNA and the ribosome.     

Helix 69 is key for uniformity during substrate selection on the ribosome

Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.     

Packing them up and dusting them off: RNA helicases and mRNA storage

Cytoplasmic mRNA can be translated, translationally repressed, localized or degraded. Regulation of translation is an important step in control of gene expression and the cell can change whether and to what extent an mRNA is translated. If an mRNA is not translating, it will associate with translation repression factors; the mRNA can be stored in these non-translating states. The movement of mRNA into storage and back to translation is dictated by the recognition of the mRNA by trans factors. So, remodeling the factors that bind mRNA is critical for changing the fate of mRNA. RNA helicases, which have the ability to remodel RNA or RNA–protein complexes, are excellent candidates for facilitating such rearrangements. This review will focus on the RNA helicases implicated in translation repression and/or mRNA storage and how their study has illuminated mechanisms of mRNA regulation. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.